Abstract
Expanded potential stem cells (EPSCs) represent a distinct and developmentally primitive stem cell population characterized by their broad developmental potential, which encompasses both embryonic and extra-embryonic lineages. In this study, we employed a polycistronic cassette to directly reprogram human fibroblasts into induced Expanded Potential Stem Cells (iEPSCs). Substituting SOX2 with engineered SOX17 transcription factors resulted in an approximately five-fold increase in the average yield of iEPSC colonies, while maintaining the molecular and functional integrity of the resulting clonal lines. Notably, under feeder-free conditions, SOX2 occasionally failed to reprogram and yielded inconsistent colony numbers, whereas engineered SOX17 and miniaturized SOX17 reproducibly produced feeder-free iEPSCs. In summary, the use of engineered SOX17 enables efficient and robust reprogramming of human fibroblasts into EPSCs, allowing for modeling of early human pre-implantation development, investigating placental disorders, and expanding the toolkit for drug development with a versatile model of pluripotent stem cells that exhibit broader developmental capabilities.
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Data availability
All plasmids generated in this study are available in Addgene or can be obtained from the lead contact upon request with a completed Materials Transfer Agreement. The RNA-sequencing data were deposited with the ArrayExpress at https://www.ebi.ac.uk/biostudies/arrayexpress under the accession number E-MTAB-16152. The processed datasets of RNA-sequencing generated in this study are also available in Supplementary Data 3. All previously published RNA-seq datasets used in our analyses are publicly available1,9,20, and the corresponding accession numbers are: ArrayExpress accession E-MTAB-7253, specifically run ERR3153919, ERR3153920, ERR3153917, ERR3153918; SRA accession number SRP115256, specifically run SRR6001369, SRR6001370, SRR6024552, SRR6026885, SRR6049361, SRR6049358, SRR6024552, SRR6026885, SRR6007374, SRR6007372, SRR6036267, SRR6037375; GEO accession numbers GSE30567, specifically run SRR307903, SRR307904. The source data underlying the graphs in the main figures and Supplementary Figs. can be found in Supplementary Data 2. We have uploaded our step-by-step protocol for iEPSC reprogramming at protocol.io with https://doi.org/10.17504/protocols.io.6qpvrynrzgmk/v1. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
Code availability
The scripts used in this study for bulk RNA‑sequencing and reprogramming data processing are available upon request.
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Acknowledgements
This study was supported by the InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government. This research was funded by the Research Grants Council of Hong Kong General Research Fund [RGC/GRF, 17106622, 17117925, and 17101120 to R.J.]. We acknowledge support and encouragement from Revive & Restore and the Applied Stem Cell Conservation Fund. We gratefully acknowledge Pik Fan Wong and the CTSCB administrative team for their support, and Yiyi Xuan for providing the SNL76/7 and SNL76/7-P cell lines.
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Conceptualization, H.H., D.H.H.H., D.R., and R.J.; methodology, H.H., D.R., D.H.H.H., Y.T., S.Y.H., M.W., and R.J.; software, H.H., M.W.; investigation, H.H., and S.W.Y.; writing – original draft, H.H., S.Y.H., and R.J.; writing—proofreading, H.H., S.W.Y., D.R., D.H.H.H., Y.T., S.Y.H., M.W., and R.J.; funding acquisition, R.J.; resources, R.J.; supervision, R.J.
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The authors declare the following competing interests: H.H., D.H.H.H., M.W., and R.J. are inventors on U.S. Patent Application No. 18/453,112 (published as US 2024/0084265 A1) relating to this work. The authors may be entitled to royalty or other financial benefits under their institutions’ IP policies. All other authors declare no competing interests.
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Hu, H., Ho, D.H.H., Yeung, S.W. et al. Enhancing reprogramming towards induced human expanded pluripotency through substitution of SOX2 with engineered SOX17 transcription factors. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09963-7
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DOI: https://doi.org/10.1038/s42003-026-09963-7
